Nile red solution

Lipid content in embryos was determined as previously described [50 (link)]. Briefly, putative zygotes (day 1) and expanded blastocyst (days 7 and 8) were collected and washed three times in PBS-PVP. At least two embryos were set aside for negative controls, and the remaining embryos were fixed as described before. Negative control embryos were incubated in 100 μl of 100% ethanol for 30 min to dissolve the lipids present. Following fixation, the remaining embryos were placed into a droplet of Nile Red Solution (Invitrogen Molecular Probes) for 30 min (1 μg/ml dilution). After incubation, embryos were washed with PBS-PVP three times and then nuclei were stained with Hoechst 33342 (1 μg/ml dilution) for 20 min in the dark. Samples were mounted on slides in Slow fade® Gold antifade reagent (Thermo Fisher Scientific) and analyzed by using fluorescence microscopy. Excitation/emission was 350/461 for Hoechst 33342 and 552/636 for Nile Red. ImageJ Software 1.46r (National Institutes of Health) was used to quantify mean intensity units per embryo to estimate the amount of lipids. A total of 230 zygotes (109 control, 120 +FLI) and 223 blastocyst stage embryos (116 control, 106 +FLI) were analyzed across 4 replicates in each experiment.